Specific Aim 1: The roles of syntabulin in neuronal transport and activity-dependent presynaptic plasticity [unreadable] [unreadable] Neuronal transport includes the intracellular trafficking route for membranous protein carriers from the soma to nerve terminals where they deliver cargos for synapse formation. The contents of these transport packets include protein components of SV and AZs, exocytotic machinery, channels, and adhesion molecules. Cargos must be attached to their transport motors with a high degree of specificity to preserve cargo identity and targeted trafficking. However, the mechanism underlying motor-cargo interactions remains unresolved. The SNARE protein syntaxin-1, a key component of SV fusion machinery, is transported to the plasma membrane in cargos. We identified a novel syntaxin-binding and kinesin-1 motor (KIF5)-associated protein named syntabulin (1, 2). Our studies with loss-of-function analysis established that syntabulin is an adaptor capable of conjoining syntaxin-1 and KIF5 motors, thereby mediating transport of syntaxin-1 to neuronal processes. [unreadable] [unreadable] Remodeling of pre-existing synapses and the formation of new synapses play an important role in the various forms of synaptic plasticity of complex neuronal networks. Previously identified mechanisms underlying activity-dependent synaptic plasticity include activation of transcriptional factors, new protein synthesis, and reorganization of the actin filaments at synapses. Thus, efficient and targeted axonal transport of newly synthesized synaptic components to presynaptic boutons would be critical in response to neuronal activity. However, the mechanisms that underlie the contribution of the microtubule-based axonal transport to the activity-induced formation of new synapses are unknown. Since syntaxin-1 is a component of the AZ precursor cargos, further characterization of syntabulins role in neuronal trafficking will contribute to understanding the molecular mechanisms of the axonal delivery of presynaptic components. Our ongoing research reveals that syntaxin-1, syntabulin, and KIF5 comprise the transport machinery critical for anterograde axonal transport of the AZ precursors and contributes to presynaptic assembly (3). Knockdown of syntabulin or disruption of the syntaxin-1-syntabulin-KIF5B complex impairs the anterograde transport of AZ components out of the soma and reduces the axonal densities of SV clusters and FM4-64 loading. Furthermore, syntabulin loss-of-function results in a reduction in both the amplitude of postsynaptic currents and the frequency of asynchronous quantal events, and abolishes the activity-induced recruitment of new AZ components into the axons and subsequent co-clustering with SVs. Consequently, syntabulin loss-of-function blocks the formation of new presynaptic boutons during activity-dependent synaptic plasticity. These studies establish for the first time that a kinesin motor-adaptor complex is critical for the anterograde axonal transport of AZ components, thus contributing to activity-dependent presynaptic assembly during neuronal development.[unreadable] [unreadable] To explore the function of syntabulin in vivo, we will generate the conditional KO mice with functional disruption of the syntabulin gene. Phenotype analysis of the KO mice and the mutant neurons will clarify the molecular details of how this protein mediates the trafficking of the AZ precursor vesicles essential for presynapse assembly and synaptic plasticity in developing neurons and brain slides.[unreadable] [unreadable] Specific Aim 2: The roles of Snapin in neurotransmitter release and synaptic modulation [unreadable] [unreadable] In neurons and neurosecretory cells, the fusion of SVs or large dense core vesicles (LDCVs) with the plasma membrane results in the release of neurotransmitters. Ca2+-triggered exocytosis depends on the presence of a pool of primed release-ready vesicles. The priming step corresponds to the assembly of the SNARE complex, in which the vesicle-associated VAMP interacts with plasma membrane-associated SNAP-25 and syntaxin-1. Maturation into a release-ready vesicle requires synaptotagmin-I, which provides Ca2+-dependent regulation of the fusion machinery. New evidence has emerged indicating that the interaction of synaptotagmin with SNAP-25 or the SNARE complex is critical for SV release and provides a clue as to how a calcium sensor is structurally and functionally coupled to the SNARE-based fusion machinery. However, the mechanisms underlying regulation of this coupling during the vesicle priming remain unclear. [unreadable] [unreadable] We initially identified Snapin as a SNAP-25 binding protein that enhances the association of synaptotagmin-I with the SNARE complex (4). The physiological role of Snapin in SV exocytosis was examined and further confirmed by microinjection of Snapin into presynaptic SCG neurons in culture (4), and by over-expression of Snapin in adrenal chromaffin cells (5) and hippocampal neurons (6). To critically evaluate the physiologic role of Snapin, we generated snapin knockout (KO) mice, which effectively avoids potential problems caused by using recombinant proteins and peptides in neurons. Our studies using the snapin KO mice in combination with genetic rescue experiments provide evidence that Snapin modulates neurosecretion in chromaffin cells by stabilizing the structural coupling of synaptotagmin to the SNARE complex, a critical step for priming docked vesicles for fusion (7). The deletion of Snapin leads to a marked reduction in the amount of synaptotagmin-I-SNARE complex. Exocytosis of LDCVs in snapin (-/-) chromaffin cells displayed a selective reduction in the exocytotic burst without a change in the sustained component of release, suggesting a reduction in the pool size of primed release-ready vesicles. The observed phenotypes can be fully rescued by re-introduction of the snapin gene into the mutant cells. However, several important questions remain to be addressed in our current studies: (1). How does Snapin regulate the interaction of synaptotagmin-I with the SNARE complex? (2) Does Snapin play a role in SV priming at CNS synapses? These studies will provide a full evaluation of the role of Snapin in SV release at central synapses, and by extension, reveal the molecular and physiological details of how this protein modulates synaptic transmission. [unreadable] [unreadable] Lab papers published related to the project:[unreadable] 1. Qingning Su*, Qian Cai*, Claudia Gerwin, Carolyn L. Smith, Zu-Hang Sheng (2004). Nature Cell Biology 6, 941-953. [unreadable] 2. Qian Cai, Claudia Gerwin, and Zu-Hang Sheng. (2005). Journal of Cell Biology 170, 959-969.[unreadable] 3. Qian Cai, Pingyue Pan, and Zu-Hang Sheng. (2007). Journal of Neuroscience 27, 7284-7296. [unreadable] 4. Jeffrey M. Ilardi, Sumiko Mochida, and Zu-Hang Sheng (1999). Nature Neuroscience 2, 119-124.[unreadable] 5. Milan G. Chheda, Uri Ashery, Pratima Thakur, Jens Rettig, and Zu-Hang Sheng (2001). Nature Cell Biology 3, 331-338.[unreadable] 6. Pratima Thakur, David R. Stevens, Zu-Hang Sheng and Jens Rettig (2004), Journal of Neuroscience 24, 6476-6481.[unreadable] 7. Jin-Hua Tian, Zheng-Xing Wu, Michael Unzicker, Li Lu, Qian Cai, Cuiling Li, Claudia Schirra, Ulf Matti, David Stevens, Chuxia Deng, Jens Rettig, and Zu-Hang Sheng (2005). Journal of Neuroscience 25, 10546-10555.